One of the major and ongoing challenges in clinical medicine is the accurate detection of disease at sensitivities far higher than that currently achievable. However, in order to improve detection threshold and precision, new sensing strategies are necessary. Fluorochromes already play an essential role in many biological assays, microscopy and imaging and cytometry. A key enabler has been to create fluorescent molecules that change brightness or color when interacting with their target. Unfortunately, one problem for in vivo use has been the relatively modest increase in fluorescence. In a bid to address this, we recently discovered fluorescent turn-on probes that use 'through- bond energy transfer' (TBET) to achieve >1,500-fold amplification of signal; this is more than 100 times higher than that described previously using bioorthogonal reactions (Angew Chem Int Ed, 2013.; PMID 23712730). The goal of this application is to thus now advance this cutting-edge broadly applicable detection platform to single cell imaging, sensing and analysis. To do this, we will initially synthesize and validate a small library of new agents and compare them to the most promising compounds already synthesized. Specifically, we intend to use the detection platform to perform i) perform single cell resolution intravital and intraoperative imaging for cancer detection and ii) a clinical trial to investigate the efficacy of the system for detecting cancer clls in diagnostic specimens. Beyond cancer detection, we anticipate the technology will also be broadly applicable to other pressing healthcare challenges such as testing the distribution and binding of novel drugs, detecting drug resistance, and/or performing experimental imaging using intravital microscopy.